Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO₄-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO₄ treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO₄-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.

Objective: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with hyperlipidemia, which is closely related to high levels of sugar and fat. β-sitosterol is a natural product with significant hypolipidemic and cholesterol-lowering effects. However, the underlying mechanism of its action on aquatic products is not completely understood. Methods: A high-fat diet (HFD)-induced NAFLD zebrafish model was successfully established, and the anti-hyperlipidemic effect and potential mechanism of β-sitosterol were studied using oil red O staining, filipin staining, and lipid metabolomics. Results: β-sitosterol significantly reduced the accumulation of triglyceride, glucose, and cholesterol in the zebrafish model. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that differential lipid molecules in β-sitosterol mainly regulated the lipid metabolism and signal transduction function of the zebrafish model. β-sitosterol mainly affected steroid biosynthesis and steroid hormone biosynthesis in the zebrafish model. Compared with the HFD group, the addition of 500 mg/100 g of β-sitosterol significantly inhibited the expression of Ppar-γ and Rxr-α in the zebrafish model by at least 50% and 25%, respectively. Conclusions: β-sitosterol can reduce lipid accumulation in the zebrafish model of NAFLD by regulating lipid metabolism and signal transduction and inhibiting adipogenesis and lipid storage.

Grass carp (Ctenopharyngodon idella), a globally important aquaculture species, exhibits protein-dependent growth plasticity, requiring genetic improvement for sustainable production. This study integrates genome-wide association analysis (GWAS) and genomic selection (GS) to unravel the genetic architecture of four growth traits, body weight, body length, body height, and body depth, in grass carp fed with diets of varying protein levels (20%, 25%, 30%, and 35%). Using a 21K liquid SNP array, we identified 62,736 high-quality SNPs across 24 chromosomes, with 90 SNPs significantly associated with growth traits. Notably, three SNPs (SLG14_24417024, SLG14_24417039, SLG24_30276273) exhibited pleiotropic effects on multiple traits. Functional annotation of 276 candidate genes near significant SNPs revealed enrichment in keratinocyte development, septin cytoskeleton organization, and heat acclimation pathways. Genomic prediction achieved accuracies up to 0.79 for body weight traits using 1533 optimal markers. This study provides the first comprehensive SNP resource for grass carp growth traits with different dietary treatments, bridging GWAS and genomic prediction to accelerate marker-assisted selection. Our findings not only advance genetic breeding strategies but also inform protein diet optimization, minimizing economic and environmental costs in aquaculture.

The grass carp (Ctenopharyngodon idella) is highly susceptible to infections caused by Aeromonas species, particularly A. hydrophila and A. veronii. However, the immunological mechanisms underlying co-infection by these pathogens remain largely uncharted. This study investigated the pathogenesis and host immune response in grass carp following concurrent infection with A. hydrophila and A. veronii. Mortality was observed as early as 24 h post-infection, with cumulative mortality reaching 68%. Quantitative analysis demonstrated significantly elevated bacterial loads in hepatic tissue at 3 days post-infection (dpi). Histopathological evaluation revealed severe hepatic lesions characterized by cellular necrosis, cytoplasmic vacuolization, and hemorrhagic manifestations. Comparative transcriptomic analysis of hepatic tissues between co-infected and control specimens identified 868 and 411 differentially expressed genes (DEGs) at 1 and 5 dpi, respectively. Gene ontology and KEGG pathway analyses revealed significant enrichment of immune-related genes primarily associated with Toll-like receptor signaling and TNF signaling cascades. Notably, metabolic pathways showed substantial suppression while immune responses were significantly activated after infected. These findings provide novel insights into the host–pathogen interactions during Aeromonas co-infection in grass carp, which may facilitate the development of effective prevention and control strategies.

Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA–mRNA–protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.